Formulations comprising vitamin b12, method of production and use thereof

ABSTRACT

Particles comprising a vitamin B12-containing microbial biomass and a solid carrier and compositions comprising said particles and a method for the production of such particles and compositions.

FIELD OF THE INVENTION

The present invention relates to particles comprising a vitaminB12-containing microbial biomass and to compositions comprising thesame. The invention further relates to a method for producing suchparticles and compositions comprising said particles. The invention alsorelates to animal feed, human food or food supplements comprising saidparticles.

BACKGROUND OF THE INVENTION

Microorganisms are known as valuable sources of a varied range of usefulcompounds. Several of these compounds are located either inside or areassociated with the microbial cell. Generally, to recover such compoundsafter fermentation of the microorganisms, it is necessary to separatethe compound from the microbial biomass. However, often such compoundsare unstable to isolation techniques or when the used microorganisms aremicrobiologically safe and food-grade, the compounds are not produced inisolated form but are produced in dry formulation together with thebiomass of the organism in which they are produced. Such formulationsare especially suited for use as animal feed supplement. By “microbialbiomass” we mean a microorganism-containing product resulting fromfermentation, which consists of whole, preferably non-viable cells (i.e.dead or killed) and/or cell debris (e.g. broken/disintegrated/lysed cellwalls).

Vitamin B12 is an important compound for humans and animals and it is animportant animal feed supplement as growth enhancer. The term “vitaminB12” is used to describe compounds of the cobalt corrinoid family, inparticular those of the cobalamin group. In this specification the term“vitamin B12” means all the cobalt corrinoids of the cobalamin group,which include in particular cyanocobalamin, hydroxocobalamin,methylcobalamin, 5′-adenosylcobalamin and 5′-desoxyadenosylcobalamincharacterised by cyano, hydroxyl, methyl or 5′-desoxyadenosyl radical(s)respectively. The methylcobalamin and 5′-desoxyadenosylcobalamincompounds are known to be unstable to light in isolated form and areeasily degraded to hydroxocobalamin in aqueous solution. For thisreason, commercial vitamin B12 preparations consist of the more stablecyanocobalamin.

Vitamin B12 is often obtained in industrial fermentation methods usingmicroorganisms known to produce vitamin B12.

A suitable method for the production of vitamin B12 via fermentation isdescribed in International Patent Application WO00/37699. This documentdescribes a non-continuous fermentation method for the production ofvitamin B12 wherein a strain of Propionibacterium is cultured in twodifferent fermentors under anaerobic and aerobic conditions respectivelyin a “fill and draw” fashion. The inhibiting effect of propionic acid ongrowth of Propionibacterium in the anaerobic phase can be considerablyreduced, leading to increased biomass and increased vitamin B12production at the end of the fermentation.

International Patent Application WO98/06868 describes a method for thepreparation of compositions comprising vitamin B12 in a concentration(based on dry matter) higher than 0.1% w/w. Such compositions areproduced by a method wherein microbial cells are cultured to(intracellularly) produce vitamin B12, after which the cells arepartially lysed and/or damaged to cause release of vitamin B12 into themedium. After separation of microbial biomass from the vitaminB12-comprising liquid phase, and concentration of the latter, theresulting concentrate solution and the microbial biomass can be combinedin different ratios and the resulting mixture(s) spray-dried. By thismethod, a spray-dried biomass can be obtained with a high concentrationof vitamin B12.

Even though the production of biomass with a high concentration ofvitamin B12 is advantageous in several ways (ease of transportation andconsequent reduction of costs) a high concentration in vitamin B12 canbe less desirable in some applications.

Spray-dried biomass can for example be used in animal feed. Prior to usein the production of feed, a lowering of the vitamin B12 concentrationin a biomass with high concentration of vitamin B12 may be necessary.The latter is especially desirable when lower dosages of vitamin B12 inthe animal diet (e.g. for poultry) are required. A possible solution tothis problem could be to blend the spray-dried biomass with a solidcarrier in order to reduce the vitamin B12 concentration prior to mixingwith other feed components. The blends of vitamin B12-containing biomassand solid carrier could be added to other feed components, eitherdirectly or in the form of a premix, which also contains other vitamins,minerals and/or bioactive ingredients, in order to produce the finalfeed. In order to assure good distribution of vitamin B12 in the finalfeed compositions, especially when low dosages need to be applied, it isvery important that the blends of solid carrier and vitaminB12-containing biomass are homogeneous.

Unfortunately, the inventors have observed that blends obtained bymixing spray-dried biomass and solid carriers are usually inhomogeneous.In addition, they are generally electrostatic, dusty and notfree-flowing. Some of these problems can cause problems during handlingof such blends on an industrial scale. Free-flowing characteristicscould be increased by addition of inorganic solid carriers like silica,but this does not improve homogeneity of the blends. Moreover, someinorganic solid carriers, like silica, may be dusty, hazardousespecially if inhaled, and not very desirable ingredients for animalfeed. Lack of homogeneity of these blends is undesirable as it can leadto inaccurate dosage of the vitamin B12 into the final premix and/orfeed, with unequal distribution of the nutrient between differentanimals. The latter is especially disadvantageous for smaller animalslike poultry. Thus there is a need for improved vitamin B12formulations, in particular for use in animal feed.

DESCRIPTION OF THE INVENTION

Accordingly the present invention is concerned with providing improvedformulations of vitamin B12-containing microbial biomass and a solidcarrier where the above-mentioned problems can be at least mitigated, ifnot overcome.

The present inventors have found that when vitamin B12-containingmicrobial biomass and a solid carrier are present in the same particle,blending of vitamin B12-containing biomass with a solid carrier prior tomixing with the other feed components can become superfluous, and someof the problems related to the prior art blends can be overcome.

Therefore a first aspect of the invention provides a particle comprisinga vitamin B12-containing microbial biomass and a solid carrier.

Particle Structures

The particles of the invention may have (3 or more) differentmorphologies (or structures). For instance, one possible morphology ofthe particle may be one in which the solid carrier is mainlyconcentrated near the centre of the particle while the vitaminB12-containing microbial biomass constitutes a sort of continuous filmof coating material around it. This is the preferred structure. Theparticle may have a core or central portion comprising the solid carrierand a coating (or outer layer) comprising the biomass.

A second possibility is the reverse of the first. It may be one in whichdistribution of respectively vitamin B12-containing microbial biomassand solid carrier is reversed, i.e. the biomass is concentrated in thecentre of the particle while the solid particles are more on theoutside. Thus the particle may have a core or central positioncomprising the biomass and a coating, or outer layer comprising thecarrier.

A third possibility may be one in which the particle is actuallyconstituted by a matrix of vitamin B12-containing microbial biomass inwhich particles of solid carrier are entrapped or vice-versa.

Particle Sizes

The size of the particles may vary, being preferably from 0.2 μm to 2000μm. The particle size may be from 10 μm to 1000 μm, preferably comprisedbetween 20 μm to 500 μm, even more preferably from 50 μm to 300 μm, mostpreferably from 50 μm to 150 μm.

Preferably, the particles of the invention have a (substantially)homogeneous particle size distribution. The phase “homogeneous particlesize distribution” is intended to mean that the overall particle sizedistribution can be relatively narrow, such that at least 70% w/w of theparticles, preferably at least 80% w/w, and more preferably 90% w/w ofthe particles have a particle size comprised between 20 and 500 μm, morepreferably comprised between 50 μm and 300 μm.

The particles of the invention may have a vitamin B12 concentration oftypically about 0.05%-5% w/w, more typically 0.1 to 4% w/w, 0.1 to 3%w/w, 0.1 to 2% w/w or 0.1-1% w/w, usually not exceeding 10% w/w.

Typically the moisture content of the particles is comprised between5-10% w/w such as 6 to 8% w/w or 7 to 10% w/w.

The invention further provides a composition comprising particlesaccording to the invention.

The particles, or a composition (essentially consisting) of particlesaccording to the invention may have a (substantially) homogeneousdistribution of vitamin B12 on or in the solid carrier. The particlesmay be free flowing and/or not dusty. It may be (substantially)non-electrostatic (for example, they may not stick to glass). Theparticles can be produced very economically.

The “vitamin B12-containing microbial biomass” is generally amicro-organism-containing-product resulting from fermentation ofmicroorganisms capable of producing vitamin B12 and cultured underconditions conducive thereof. It can mean a biomass (either alive ordead) comprising cells that comprise vitamin B12, such as cells thatproduce (or have produced) vitamin B12. Themicro-organism-containing-product consists of preferably non-viable(e.g. dead), whole (or intact) cells and/or cell debris comprisingvitamin B12.

Vitamin B12-containing (or producing cells), or microbial biomass,preferably comprises a bacterial strain, such as of the genusAcetobacterium, Acetobacter, Agrobacterium, Alcaligenes, Arthrobacter,Azobacter, Bacillus, Clostridium, Corynebacterium, Escherichia,Eubacterium, Flavobacterium, Methanobacillum, Methanosarcina,Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium,Rhodopseudomonas, Salmonella, Serratia, Streptococcus, Streptomyces orXanthomonas. Preferably a bacterium is used which is safe forconsumption by humans, e.g. GRAS, and/or animals. In particular, thebacterium preferably does not produce endo- or exotoxins.Propionibacteria in particular are often food-grade and satisfy thesecriteria.

Therefore in a preferred embodiment of the invention the particle ischaracterised in that the vitamin B12-containing microbial biomass isfrom the genus Propionibacterium.

Preferred Propionibacterium species used at this regard are P.freundenreichii, P. theonii, P. jensenii, P. shermanii and P.acidipropionici. In one embodiment it is preferred that a singlebacterial strain is present.

Solid Carrier

The solid carrier used in the particles of the invention may be aparticulate material or powder which is preferably non-hygroscopic. Thecarrier can be suitable for use in a spray-drying, multi-stage and/orfluid bed drying techniques. The carrier may be edible or digestible(either by animals and/or humans). The carrier will exclude cells orparts thereof.

The solid carrier in the particles according to the invention preferablyhas low bulk density. This may allow them to be used in theabove-mentioned drying techniques. Preferably the density is comprisedbetween 400 and 1200 kg/m3, preferably between 400 and 1000 kg/m3, morepreferably about 500 kg/m3. Preferably the particle size of the carrieris equal to or lower than 500 μm, preferably equal to or lower than 300μm, generally comprised between 10-300 μm, more preferably comprisedbetween 10-200 μm, most preferably comprised between 30-150 μm.

Generally the solid carrier has a moisture content of 2-15% w/w a powderfor example, 5 to 10% w/w such as 6, 8, 12 or 14% w/w.

Preferably the solid carrier comprises a carbohydrate, a protein, or amixture thereof. Suitable solid carriers comprise (powders of) casein,whey, milk, maltodextrin, corn steep solids, starch, edible flour, ormixtures thereof. Most preferably the solid carrier comprises edibleflour. With “edible flour” it is intended to cover a finely ground meal(essentially consisting of starch and protein) obtainable from ediblecereal grains or seeds (e.g. wheat, rice, maize, barley, oat, rye,etcetera), from legumes (e.g. beans, peas, etcetera) or from edibletubers or fruits such as potatoes or bananas, or a mixture thereof.Edible flour can have the advantage of being cheap, light and of being adesirable component in animal feed.

In one embodiment it is preferred that the solid carrier is not a (e.g.crystallisable) sugar such as lactose, saccharose, dextrin or othermaltodextrin. In a preferred embodiment, the particles of the inventionare characterised in that the weight ratio between the vitaminB12-containing microbial biomass and the solid carrier is between 0.2-5,preferably between 0.25-4 or 0.3-3, more preferably between 0.5-2.

The particles according to the invention can be produced according toany method suitable to the formation of composite particles, likespray-drying, fluid bed drying, multi-stage drying. Spray-drying, fluidbed drying and multi-stage drying techniques are known to those skilledin the art.

Spray-drying

Preferably the particles of the invention are produced by spray-dryingor multi-stage drying techniques. For example, a liquid (suspension of)vitamin B12-containing microbial biomass can be spray-dried in thepresence of a solid carrier (preferably in powder form). The termsspray-drying or spray-dryer are used in a broad sense, to cover bothpure spray-drying or spray dryer and multi-stage drying or multi-stagedryer.

Accordingly the invention in a second aspect provides a method for theproduction of particles comprising vitamin B12-containing microbialbiomass and a solid carrier. At its broadest, the method of the secondaspect comprises co-spray drying the biomass and solid carrier. In otherwords, the biomass and solid carrier are both spray driedsimultaneously, and preferably in contact with one another.

The spray drying of both of the biomass and the solid carrier can resultin the particles of the first aspect. Preferably, the particles willcomprise a central or core portion of the sold carrier, and a coating orouter layer of the biomass. The biomass and solid carrier willpreferably be supplied to the spray-dryer in separate streams, orthrough different ports or inlets. Suitably, the solid carrier andbiomass are only (and first) mixed once inside the spray-dryer.

A solid carrier may be in solid form, such as a powder, or in a liquidform, which is a slurry. The biomass is preferably in a liquid form,preferably a liquid suspension. The biomass (such as in the form of aliquid) may have been subjected to several processing steps prior tobeing co-spray dried with the solid carrier. It may have been subjectedto concentration and/or evaporation, dia filtration and/orpasteurisation. Thus, one or more of these steps may have been performedon a liquid comprising the biomass before spray drying occurs.Preferably, inside the spray dryer, the biomass is atomised. Atomisationmay occur before the biomass is mixed with the solid carrier.

In a preferred method, a liquid comprising (e.g. a suspension of)vitamin B12-containing microbial biomass and a solid carrier (e.g. inpowder form) are conveyed into (for example, a drying chamber) of aspray-dryer, preferably in or through separate streams or ports. Theliquid (suspension) and the solid carrier can then come into contactwith each other inside the spray-dryer chamber.

Fermentation and Pre-Spray Drying Steps

The vitamin B12-containing microbial biomass used in the method of theinvention is preferably obtainable in or from an industrial fermentationprocess using microorganisms which produce vitamin B12. These includebacteria belonging to the bacterial strains mentioned above. Severalfermentation methods suitable to the microbial production of vitamin B12are known to those skilled in the art. Preferably, the vitaminB12-containing microbial biomass is obtainable from a bacterial strainof the genus Propionibacterium. Several methods are known in the art forthe fermentation of Propionibacterium strains under conditions conduciveto the production of vitamin B12. An example is described inInternational Patent Application WO00/37699.

Generally the microbial cells containing vitamin B12 are concentratedand optionally purified at the end of the fermentation by one or moremethods suitable to this purpose (e.g. evaporation, ultrafiltration,diafiltration, etc.).

Preferably a concentration (based on dry matter) of microbial biomass inthe liquid suspension is obtained which allows economical spray-dryingoperation. In a preferred embodiment the liquid (suspension of) vitaminB12-containing microbial biomass used in the method according to theinvention has a concentration of 50-300 g/l, preferably 100-300 g/l,more preferably 200-300 g/l or 250-300 g/l based on dry mass per litreof liquid (i.e. concentrate).

Optionally it is possible to use a vitamin B12-containing microbialbiomass with a relatively low concentration, for example of about120-150 g/l, and further concentrate the liquid suspension, for exampleup to about 200-300 g/l, 220-300 g/l or 250-300 g/l, just prior tospray-drying. A concentrator/evaporator positioned upstream to thespray-dryer can be used for this purpose. Thus, a fermentation broth canbe subjected to concentration and/or evaporation to form the liquid tobe co-spray dried with the solid carrier.

Acids

It is known that carboxylic acids with a low molecular weight likeacetic and propionic acid are produced during fermentation ofPropionibacterium strains. The latter can pose a problem as the presenceof acids during spray-drying of the corresponding microbial biomass cancause difficulties in the drying process and can cause stickiness of theend product. Therefore, the liquid (e.g. suspension of Propionibacteriummicrobial) biomass is preferably treated, prior to spray-drying or multistage drying, for example by diafiltration. This may reduce the acidconcentration to a desirable value.

Optionally the vitamin B12-containing microbial biomass is pasteurisedprior to spray-drying.

Spray-dryer

The method for the production of the particles according to theinvention can be performed on a conventional spray-dryer or multi stagedryer. This type of equipment is generally used in many applications,e.g. in the dairy industry.

Generally a spray-dryer (or multi-stage dryer) comprises at least adrying chamber, such as with a distribution element for atomising aliquid to be spray-dried. It may also have means for supplying (drying)gas and/or means for discharging the (spray-dried) product from thedevice. Besides the above-mentioned elements, a multi stage-dryerfurther comprises one or more fluidised beds. A spray-dryer ormulti-stage dryer suitable for use in the method according to theinvention may further comprise means suitable to supply the solidcarrier into the drying chamber.

Therefore a spray-dryer (or multi-stage dryer) suitable for use in themethod of the invention can comprise at least two (product)-inlet ports,generally positioned on the upper part of the spray-dryer chamber.Through one inlet port the liquid (suspension of) microbial biomass canbe atomised and conveyed into the spray-dryer chamber. Said inlet portis furnished with means suitable to atomise the liquid such as anatomiser (e.g. nozzle, rotating disk atomisers etc.). A secondproduct-inlet port can be used to convey the solid carrier, (generallyin powder form) into the drying chamber. A slurry of the carrier canalso be applicable. Optionally the spray-dryer may comprise means forthe recovery of fine particles. Said (fine) particles can bereintroduced into the drying chamber by means of a third product-inletport or into the pipeline conveying either the microbial biomass or thesolid carrier into the system or dyer. Optionally, the spray-dryer ispart of a multi-stage dryer comprising one or more fluidised beds.

Typically an inlet temperature of the air in the drying chamber of thespray-dryer is used which is between 120-250° C., preferably between160-220° C. such as 180 to 200° C. The outlet temperature of the air isgenerally comprised between 60-95° C. for example 60-90° C. or 70-80° C.

Both streams of solid carrier and (atomised) liquid (suspension of)microbial biomass are conveyed into the drying chamber. A stream ofdroplets can be produced by atomisation of the liquid (suspension ofmicrobial biomass). The (e.g. atomised) liquid may then be brought intocontact with the solid carrier (e.g. in powder form). This usuallyhappens inside the drying chamber. One can then produce particlescomprising vitamin B12-containing microbial biomass and the solidcarrier.

Ratio of Biomass to Carrier

The method of the invention advantageously allows adjustment of theamount of vitamin B12-containing microbial biomass and/or (on) the solidcarrier, in order to assure an optimal distribution of vitamin B12containing biomass on the particle. Advantageously the invention alsoallows adjustment of the amount of vitamin B12 on the solid carrier,depending both on the content of vitamin B12 in the microbial biomassand on the final application of the resulting particles.

Preferably the weight ratio between the vitamin B12-containing microbialbiomass and the solid carrier used in a method of the invention isbetween 0.2-5, preferably between 0.25-4 or 0.3-3, more preferablybetween 0.5-2.

The method can be used to produce particles having the propertiesdescribed above. In a preferred embodiment the invention relates toparticles obtainable by the method. Such particles can have a number ofadvantages compared to e.g. particles formed by mixing spray-driedbiomass and solid carrier, or formed by spray drying a mixture ofbiomass and solid carrier. For example, the particles generally have ahomogeneous mean particle size distribution. The particles usually havevisual homogeneity i.e. no separation can usually be observed betweenmicrobial biomass and solid carrier. Particles obtained in this way arealso generally less hygroscopic, free flowing, less dusty and/or morefree of moulds and bacterias. The particles are particularly useful whena lower concentration of vitamin B12 is desirable e.g. for use in animalfeeds.

One advantage related to the method of the invention is that particleswith a homogeneous mean particle size distribution can be obtained.Another advantage in the method according to the invention is that thespray-drying step allows production of “pasteurised” compositionscomprising the particles according to the invention. This is especiallyadvantageous when certain types of solid carrier, which are not alwaysfree of microorganisms and yeast (e.g. edible flour) are used.

The invention thus provides particles comprising a vitaminB12-containing microbial biomass and a solid carrier obtainable by amethod of the invention. Said particles have the desirablecharacteristics already described above. The invention in a third aspectprovides compositions comprising the particles of the first aspect orparticles preparable by the second aspect. Preferred features and/orcharacteristics of one aspect of the invention are applicable to anotheraspect mutatis mutandis.

Animal Feed

The particles of the invention can be used as or in the production ofanimal feed. To this end, the particles containing vitamin B12 are addedto other feed components, either directly or in the form of a premix,which may also contain other vitamins, enzymes, minerals and/orbioactive ingredients.

Therefore the invention also provides an animal feed comprisingparticles according to the invention.

Feeding an animal a diet comprising a feed according to the inventionpromotes its growth.

Thus the invention also provides the use of an animal feed according tothe invention to promote the growth of an animal.

A further aspect of the invention relates to a premix or additivecomposition to be added to one or more edible feed substance(s) oringredient(s), for example to prepare (or for supplementation of) a feedcomposition. This can comprise the particles of the fist aspect orpreparable by the method of the second aspect. The premix can be“diluted” by a factor of 10 to 1,000 (so that the premix constitutes 10%to 0.1% of final feed) when making the animal feed. This premix may bein the form of granules or pellets.

The invention also relates to a process for the preparation of an animalfeed composition, the process comprising adding to (or supplementing) ananimal feed, or to one or more edible feed substance(s) oringredient(s), the particles of the invention.

Another aspect of the invention relates to a process for promotinggrowth, feed conversion or antibacterial activity, in a monogastric ornon-ruminant animal, the process comprising feeding the animal particlesof the invention.

Suitable animals include farm, monogastric and/or non-ruminant animalssuch as pigs (or piglets), poultry (such as chickens and turkeys),calves, veal calves or aquatic (e.g. marine) animals, for example fish.

The compositions of the invention, in particular additive or premixcompositions, can be either in liquid or solid form. If a solid, thenthis may be a powder, a granulate, extrudate or it may be pellets. For asolid form, the amount of water present may be below 20, 15 or even 10%,such as from 2 to 10%, 3 to 8% or 4 to 7%.

The remainder may comprise carbohydrates and/or carbohydrate polymers(such as starch and/or modified starch), for example at least 70, 80, 90or 95%, such as from 75 to 90%. The composition may have a coating, forexample if it is in a pellet, granulate, or extrudate form. There maythus be one or more coats on the outside of the composition, comprisingone or more coating materials. If present, the coating (or coatingmaterials) may be present at from 1 to 10%, such as from 2 to 6%,optimally at from 3 to 5%. The composition may have one or morestabilisers (such as glycerol and/or sorbitol) and/or one or morepreservatives (such as sorbate and/or benzoate).

If the composition is a liquid, then the water (or moisture) contentwill be higher. The water content may be up to 40, 50 or 60%, forexample from 25 to 65%, optimally from 35 to 55%. If a stabiliser ispresent, this may be at an amount of from 45 to 65%, such as from 50 to60%, optimally from 52 to 58%. The stabiliser is preferably sorbitoland/or glycerol.

A description of the preparation of pellets and granules, in particularcarbohydrate based enzyme granulates, is described in WO-A-98/54980(International Application No. PCT/EP98/03327). This and all otherdocuments mentioned have their contents incorporated herein byreference.

The composition may comprise a carrier which may comprise at least 15%of an edible carbohydrate polymer. The carrier may be in particulate orpowder form. However, if the composition is a liquid, it may be in theform of a solution or a slurry. The polymer preferably comprisesglucose, or glucose-containing units, although it can containglucopyranose units, amylose and/or amylopeptin. In addition, or insteadof starch, a glucan, peptin or glycogen can be used. Preferably at least15%, such as at least 30%, at least 40%, for example at least 60%,optimally at least 80% of the composition (or the solid carrier)comprises the carbohydrate polymer.

Additional details of enzyme-containing compositions for animal feed canbe found in WO-A-98/55599 (International Application No.PCT/EP98/03328).

Animal feed compositions of the invention will usually contain one ormore feed ingredients or substances. These are ingredients andsubstances intended for consumption by an animal, and is therefore in aform suitable for ingestion and nutrition for an animal. Preferably thefeed composition is both edible and digestible by the animal.

Suitably the ingredients and/or substances have a dry matter content ofat least 80, 85, 90 or 95%. The protein content of the composition (orthe substances and/or ingredients) may vary considerably, but may befrom 5 to 20%, such as 10 to 15%, for example vegetable and/or plantproducts or parts thereof, such as buckwheat, rice, wheat, barley orcorn. Substances or ingredients with higher protein contents, such asfrom 45 to 95%, e.g. 50 to 80%, may be provided, for example peanuts,poultry feathers, soy bean (or products thereof), sunflower (e.g. seeds)or casein. Preferred animal feed compositions may therefore comprise oneor more of oats, pea (seeds), peanuts, soy beans, sunflower, canola,casein, coconut, corn, meat, millet, potato, rice, safflower and/orwheat. Preferably the composition (and substances or ingredients) have acrude fibre content below 30%, 25%, 20%, 15% or even below 10%.Similarly, the calcium content may be below 2%, such as 1%, below 0.5%and preferably less than 0.2%. The total phosphorous content of the(animal feed composition) is preferably from 2 to 0.01%, such as from 1to 0.1%, optimally less than 0.5%.

The precise substances and ingredients can vary depending on the animalto be fed. An alternative composition may comprise one or more of bakerywaste, sugar beet, brewers grain, canola, cassaya, corn, fababean, fish(such as anchovy or herring meal), lentils, meat and/or millet.

The particles of the invention can also be used in the production of ahuman food, foodstuff or food, dietary or nutritional supplement or apharmaceutical composition. Therefore the invention provides any ofthese compositions comprising particles according to the invention.

The invention will now be illustrated, by way of Examples, which are notintended to be limiting.

EXAMPLES

General Methods

Fermentation broth from Propionibacterium freudenreichii CBS 929.97 wasobtained as described in International Patent Application WO00/37699.

The fermentation broth was concentrated by means of ultrafiltration (onpolysulfon MW cut off 5-10 kD, Koch HFK 151 VSV) or microfiltration (onMembralox ceramic 0.1 μm) up to a biomass concentration of 100-150 g/l.

After ultrafiltration or microfiltration, the propionic acid in thebiomass had a concentration of about 25-30 g/l. To reduce theconcentration of propionic and acetic acid in the biomass, the biomassconcentrate was diafiltered with water. This diafiltration was performedby an in-line addition of water to the concentrate at the same rate asthe permeate flow. The diafiltration was stopped at a propionic acidconcentration lower than 5 g/l. At this purpose a ratio (v/v) water:concentrate of 3-4:1 was applied.

After diafiltration the concentrated biomass was pasteurised during 1minute at a temperature of 90-940 C (either by direct steam injection orheating by a plate heat exchanger).

The pasteurized biomass was further concentrated by a multistage(vacuum) falling film evaporator with vapor recompression. This type ofevaporator is known to those skilled in the art.

The following conditions were applied. Biomass feed rate 2000-3000 1/h(corresponding to 300 kg dry matter/h) Pre-heater temperature 920 C. 1ststage temperature 65-700 C. 5th stage temperature 50-550 C. Temperatureof concentrate 45-500 C. Biomass concentration 22-26% (1250 kg/h)

The biomass concentrate was spray-dried on a Multi Stage Dryer (NIRO AS,Denmark).

The following set up was used in all the experiments.

The vitamin B12-containing biomass was fed into the drying chamber by anozzle with a biomass feed rate of 1250 kg dry matter/h). Nozzlepressure 190-195 bar Air inlet temperature (co current) 200-2200 C. Airoutlet temperature 75-920 C. Air Internal fluid bed temperature 55-600C. Air 1st external fluid bed temperature 30-350 C. Air 2nd externalfluid bed temperature 15-200 C. Powder temperature <300 C.

Fines were returned via a cyclone to the nozzle area.

(Comparative) Example 1

In this example vitamin B12-containing biomass was spray-dried inabsence of solid carrier applying the above-mentioned spray-dryingconditions.

Example 2

In this example 600 kg of vitamin B12-containing spray-dried biomassobtained in Example 1 was mixed in an external powder mixer (batch) with300 kg of wheat flour and 1 kg of silica (Aerosil 200®).

Example 3

In this example 120 g MgSO4.7H2O per kg of concentrate was added to thediafiltered biomass concentrate (120 g/l biomass concentration) beforeevaporation. The mixture was evaporated to a dry matter content of 32%and spray dried as described above.

Example 4

In this example vitamin B12-containing biomass was spray-dried inpresence of wheat flour as a solid carrier applying the above-mentionedspray-drying conditions. The wheat flour was dosed as a powder at a rateof 180-220 kg/h Both streams of solid carrier and atomised liquidsuspension of microbial biomass were separately conveyed into the spraydryer chamber. The powder was dosed into the spray dryer chamber closeto the area of the nozzle feed stream.

Results

The characteristics of the compositions comprising vitaminB12-containing spray-dried biomass obtained in Examples 1 to 4 wereanalysed and are reported in the following table. Example 1 2 3 4Vitamin B12 content (mg/kg) 1600 1080 985 1110 Dry matter (% w/w) 94 9486 94 Presence of lumps no no yes no Dust (mg/kg) 50 10500 410 320Flowability ok ok ok ok Particles (% w/w) with particle n.r. 91 99 82size lower than 300 μm) Total plate count per g 20 14000 20 500 Mouldsper gram 100 500 10 20 Visual homogeneity yes no no yes

Visual homogeneity in the context of the present table means that thedistribution of microbial biomass on the solid carrier is visuallyhomogeneous, i.e. no separation is observed between spray-driedmicrobial biomass and solid carrier.

By comparing the results obtained in Example 4 with those obtained inthe other examples it is clear that compositions essentially consistingof the particles of the invention can be homogeneous, not hygroscopic,free-flowing, not dusty and almost free of moulds and bacteria.

1. A particle comprising vitamin B 12-containing microbial biomass and asolid carrier.
 2. A particle according to claim 1 wherein the weightratio between the vitamin B 12-containing microbial biomass and thesolid carrier is between 0.2-5 and/or the particles have been preparedby spray-drying (i.e. the particles are spray-dried).
 3. A particleaccording to claim 1 wherein the (or average) particle size is comprisedbetween 0.2 um and 2000 um, and/or particles have been prepared byco-spray drying the biomass and the solid carrier.
 4. A particleaccording to claim 1 wherein the vitamin B 12-containing microbialbiomass comprises a bacterial strain of the genus Acetobacterium,Acetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azobacter,Bacillus, Clostridium, Corynebacterium, Escherichia, Eubacterium,Flavobacterium, Methanobacillum, Methanosarcina, Mycobacterium,Propionibacterium, Proteus, Pseudomonas, Rhizobium, Rhodopseudomonas,Salmonella, Serratia, Streptococcus, Streptomyces and/or Xanthomonas. 5.A particle according to claim 1 wherein there is a core or centralportion comprising the solid carrier and a coating or outer layercomprising the biomass.
 6. A particle according to claim 1 wherein thesolid carrier comprises a carbohydrate, a protein, or a mixture thereof.7. A particle according to claim 6 wherein the solid carrier comprisescasein, whey, milk, maltodextrin, corn steep solids, starch, edibleflour, or a mixture thereof preferably as a powder.
 8. A particleaccording to claim 7 wherein the edible flour is obtainable from wheat,rice, barley, maize, oat, rye, potato, banana, or a mixture thereof. 9.A method for producing particles comprising vitamin B 12-containingmicrobial biomass and a solid carrier, the method comprising co-spraydrying the biomass and the carrier.
 10. A method according to claim 9wherein a liquid comprising the vitamin B 12-containing microbialbiomass and a solid carrier in powder form are used.
 11. A methodaccording to claim 9 wherein the biomass and carrier are conveyed into adrying chamber of a spray-dryer through separate streams or ports.
 12. Amethod according to claim 9 wherein a liquid suspension of biomass andthe solid carrier are contacted with each other inside the spray-dryer.13. A method according to claim 9 wherein the weight ratio between thevitamin B 12-containing microbial biomass and the solid carrier isbetween 0.2 and
 5. 14. A method according to claim 9, wherein the solidcarrier has a particle size equal to or lower than 500 pm.
 15. A methodaccording to claim 9 wherein the vitamin B12-containing microbialbiomass comprises, or is obtainable from a bacterial strain of the genusPropionibacterium.
 16. A method according to claim 9 wherein the solidcarrier is a carbohydrate, a protein, or a mixture thereof.
 17. A methodaccording to claim 9 wherein the solid carrier comprises edible flour.18. A method according to claim 9 wherein the liquid comprising thebiomass has been subjected to concentration or evaporation,pasteurisation and/or diafiltration prior to spray drying and/or theliquid is atomised inside the spray dryer.
 19. A method of claim 10wherein the concentration of vitamin B12-containing microbial biomass inthe liquid suspension is between 50 and 300 g/1.
 20. A particlecomprising vitamin B12-containing microbial biomass and a solid carrierobtainable by a method according to claim
 9. 21. A compositioncomprising particles according to claim
 1. 22. An animal feed comprisingparticles according to claim
 1. 23. A composition edible by humanscomprising particles according to claim
 1. 24. A composition accordingto claim 23, which is a human food or food supplement, foodstuff,nutritional or dietary supplement or a pharmaceutical composition.